Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

Abstract

Iron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

SifR is an Rrf2-family quinone sensor associated with catechol iron uptake in Streptococcus pneumoniae D39

“…We further document here that enzymes encoded by three of the most highly differentially expressed genes in a Δ sifR mutant possess the anticipated broad spectrum Fe II –catechol 2,3-dioxygenase activity ( spd_0072 ; catE ), quinone reductase activity ( spd_1440 ; ywnB ), and ferric reductase activity ( spd_1375 ; yhdA ) ( 76 , 77 ). The first two activities in combination are expected to convert oxidized catechols, brought into the cytoplasm as Fe III chelates, to the corresponding 2-hydroxy acid semialdehydes, whereas the third allows direct assimilation of ferrous Fe ( Fig.…”

Production and Potential Genetic Pathways of Three Different Siderophore Types in Streptomyces tricolor Strain HM10

“…FhuF, a ferric siderophore reductase (FSR), is involved in iron release from the siderophore inside the cell as described in E. coli. Otherwise, siderophore-interacting proteins (SIPs) perform the same process [68].…”

Extracytoplasmic Function Sigma Factors Governing Production of the Primary Siderophores in Pathogenic Burkholderia Species

“…Once in the cytoplasm, the iron must be released, which may involve hydrolysis of the siderophore by esterases to liberate Fe(III). Alternatively, Fe(III) may be reduced to Fe(II) by ferric reductases, as ferrous iron has much lower affinity for the siderophore ( Trindade et al, 2021 ).…”